姜黄素预处理的骨髓间充质干细胞外泌体对骨关节炎软骨细胞的影响

Effect of curcumin-primed human bone marrow mesenchymal stem cells-derived exosomes on osteoarthritic chondrocytes

目的观察姜黄素预处理的骨髓间充质干细胞(BMSCs)外泌体对骨关节炎软骨细胞增殖、凋亡以及关键基因、蛋白表达的影响。方法从郑州大学第一附属医院骨外科选择10例在2023年3月至2023年6月住院的患者, 在手术中提取人BMSCs(hBMSCs)和骨关节炎软骨细胞进行培养, 其中股骨颈骨折患者5例, 年龄(56.8±6.0)岁, 女3例, 膝关节骨关节炎5例, 年龄(54.6±5.9)岁, 女4例。在BMSCs培养基中加入10 μmol/L姜黄素培养48 h后, 从BMSCs培养上清中利用差速离心法和超速离心法提取姜黄素外泌体, 并对其进行鉴定。将骨关节炎软骨细胞分为对照组、白细胞介素(IL)-1β组(1 ng/ml IL-1β预刺激)、IL-1β+姜黄素组(1 ng/ml IL-1β预刺激+游离姜黄素)、IL-1β+姜黄素外泌体组(1 ng/ml IL-1β预刺激+经过姜黄素预处理的BMSCs来源的外泌体), 并用噻唑蓝(MTT)法、半胱酰胺天冬氨酸特异性蛋白酶(Caspase)-3/7活性试验检测姜黄素预处理外泌体对骨关节炎软骨细胞增殖、凋亡的影响。采用实时荧光定量聚合酶链反应技术(RT-PCR)和蛋白印迹法(Western blot)检测关键基因、蛋白的表达差异, 两组以上比较采用单因素方差分析(one-way ANOVA)。结果提取到的外泌体为粒径约90~120 nm之间的圆形膜性囊泡, 表达外泌体特异性标记物CD9、CD63。荧光显微镜下可见软骨细胞内的由红色荧光标记的外泌体。MTT法检测结果显示, 对照组设置为1.00, IL-1β组(0.50±0.03), IL-1β+姜黄素组(0.51±0.04), IL-1β+姜黄素外泌体组(0.83±0.03)。IL-1β+姜黄素外泌体组增殖能力明显高于IL-1β组(0.83±0.03比0.50±0.03, t=8.77, P<0.001), 差异有统计学意义。Caspase-3/7活性细胞凋亡检测显示, 对照组设置为1.00, IL-1β组(1.53±0.08), IL-1β+姜黄素组(1.51±0.05), IL-1β+姜黄素外泌体组(0.73±0.04)。IL-1β+姜黄素外泌体组凋亡率明显低于IL-1β组(0.73±0.04比1.53±0.08, t=11.89, P<0.001), 差异有统计学意义。RT-PCR结果显IL-1β+姜黄素外泌体组IL-6、MMP-13表达显著低于IL-1β组(0.59±0.04、0.39±0.03比1.00, 0.68±0.04、0.40±0.04比1.00, IL-1β组设置为1.00, t=25.24、22.11, P<0.01), COL2A1表达显著高于IL-1β组(2.39±0.17、2.91±0.17比1.00, t=16.19, P<0.01), 而IL-1β+姜黄素外泌体组与IL-1β+姜黄素组相比, COL2A1表达更高。结论姜黄素预处理的BMSCs外泌体能够使骨关节炎软骨细胞增殖增强, 凋亡减少, 减弱软骨基质分解, 促进软骨修复。

Objective To observe the effect of curcumin-primed exosomes derived from human bone marrow mesenchymal stem cells(hBMSCs)on osteoarthritic chondrocyte proliferation,apoptosis and expression of key genes and proteins.Methods A total of 10 patients who were hospitalized from March 2023 to June 2023 were selected from the Department of Orthopedic Surgery of the First Affiliated Hospital of Zhengzhou University,and hBMSCs and osteoarthritic chondrocytes were extracted and cultured from 10 patients undergoing joint replacement,including 5 patients with fracture of neck of femur,aged(56.8±6.0),60%females,and 5 patients with osteoarthritis of the knee,aged(54.6±5.9),80%females.After adding 10μmol/L curcumin to the BMSCs culture medium for 48 h,curcumin exosomes were extracted from the culture supernatants of the BMSCs using differential centrifugation and ultracentrifugation(100000×g centrifugation for 70 min).Osteoarthritic chondrocytes were categorized into control group,interleukin(IL)-1βgroup(1 ng/ml IL-1βpre-stimulation),IL-1β+curcumin group(1 ng/ml IL-1βpre-stimulation+free curcumin)and IL-1β+curcumin-exosomes group(1 ng/ml IL-1βpre-stimulation+curcumin-primed hBMSC-derived exosomes),and the effect of curcumin-primed hBMSC-derived exosomes on the proliferation and apoptosis of osteoarthritic chondrocytes was detected by methyl thiazolyl tetrazolium(MTT)assay,cysteinyl aspartate-specific protease(Caspase)-3/7 apoptosis assay,respectively.Real-time fluorescent quantitative polymerase chain reaction(RT-PCR)and Western blotting were used to detect differential expression of key genes and proteins.Comparison of data above the two groups was performed by one-way ANOVA.Results The extracted exosomes were round membranous vesicles with a particle size of about 90-120 nm,and expressed exosome-specific markers CD9,CD63,indicating successful characterization.Exosomes labeled by red fluorescence within chondrocytes were visible under fluorescence microscopy,indicating that exosomes were taken up by chondrocytes.The results of MTT method showed that the control group was set to 1.00,IL-1βgroup(0.50±0.03),IL-1β+curcumin group(0.51±0.04)and IL-1β+curcumin-exosome group(0.83±0.03).The proliferative capacity of the IL-1β+curcumin-exosomes group was significantly higher than that of the IL-1βgroup(0.83±0.03 vs.0.50±0.03,t=8.77,P<0.001).Caspase-3/7 activity apoptosis assay showed that the control group was set to 1.00,and the IL-1βgroup(1.53±0.08),the IL-1β+curcumin group(1.51±0.05),and IL-1β+curcumin exosome group(0.73±0.04).The apoptosis rate in the IL-1β+curcumin-exosomes group was significantly lower than that in the IL-1βgroup(0.73±0.04 vs.1.53±0.08,t=11.89,P<0.01).RT-PCR results showed that IL-6 and MMP-13 expression in the IL-1β+curcumin exosome group was significantly lower than that in the IL-1βgroup(0.59±0.04,0.39±0.03 vs.1.00,0.68±0.04,0.40±0.04 vs.1.00,set at 1.00 in IL-1βgroup,t=25.24、22.11,P<0.01),COL2A1 expression was significantly higher than that in IL-1βgroup(2.39±0.17,2.91±0.17 vs.1.00,t=16.19,P<0.01),and IL-1β+curcumin exosome group had lower expression of IL-6,MMP-13 and higher expression of COL2A1 that IL-1β+curcumin group.Conclusion Curcumin-primed hBMSC-derived exosomes enhance osteoarthritic chondrocyte proliferation,reduce apoptosis,attenuate cartilage matrix decomposition,and promote cartilage repair.